Method of removing isoflavones and phytates

ABSTRACT

Methods for sequentially removing isoflavones and phytates from an active surface by utilizing an aqueous medium for isoflavone removal and an aqueous medium for phytate removal. The aqueous medium for isoflavone removal contains at least one alcohol and at least one acid. The aqueous medium for phytate removal is either a relatively stronger acidic solution, a basic solution, or with some active surfaces may be an aqueous solution of pH 2-7, which is essentially free of alcohol and organic solvents. The use of the methods disclosed allows sequential isolation of isoflavones and phytates, compounds which may then be utilized in various foods for human consumption.

FIELD OF THE INVENTION

[0001] The invention relates to methods for sequentially removingisoflavones and phytates from active surfaces.

BACKGROUND

[0002] Plant proteins are frequently utilized as protein sources in foodfor human consumption such as nutritional formulas or cereals, but areoften purified prior to such use. Purification may be utilized to removecompounds such as phytoestrogens or plant estrogens, manganese ornucleotides. Phytoestrogens are plant substances that are structurallyand functionally similar to the gonadal steroid, 17 β-estradiol, thatproduce estrogenic effects. There are three main groups of nonsteroidaldietary estrogens: (1) isoflavones, (2) coumestans, and (3)mycoestrogens (fungal). A review of phytoestrogens and their effects inmammals is reported by Kaldas and Hughes in “Reproductive and GeneralMetabolic Effects of Phytoestrogens in Mammals,” ReproductiveToxicology, vol. 3, pp. 81-89, 1989. As used herein, the term“isoflavones” is equivalent to the term “phytoestrogens” as the term isdefined in the Kaldas et al. article. Thus, isoflavones includeflavonones, flavonols, flavones, isoflavones, aurones, chalcones,dihydrochalcones, anthocyanins, leucoanthocyanins, leucoanthocyanidins,anthocyanidins, anhydroflavenols, catechins and chemical derivatives ofthese groups.

[0003] Research has suggested that isoflavones may inhibit the growth ofhuman cancer cells. See e.g., Setchell, K. D. R., and Welch, M. B., F.Chrom., 386 (1987), pp. 315-323; High Performance Liquid ChromatographicAnalysis of Phytoestrogens in Soy Protein Preparations with Ultraviolet,Electrochemical and Thermospray Mass Spectrometric Detection, McLachlan,J. A., ed. Estrogens in the Environment, New York: Elsevier Press, 1985,pp. 69-85; and Setchell, et al., “Nonsteroidal Estrogens of DietaryOrigin: Possible Roles in Hormone Dependent Disease,” Am. F. Clin.Nutr., 1984, 40, pp.569-578. There is also some indirect, demographicsupport for an isoflavone mediated reduction in cancers of hormoneresponsive tissues based on observations that women in countriesconsuming vegetarian diets have a lower incidence of breast cancercompared to women in meat-eating countries. Adlercreutz et al.,“Determination of Urinary Lignans and Phytoestrogen Metabolites,Potential Antiestrogens and Anticarcinogens, in Urine of Women onVarious Habitual Diets,” Steroid. Biochem., 1986, 25, pp. 791-797.Isoflavones have also been suggested to have antiviral and fungicidalproperties. And, they have been implicated in the reduction of serumcholesterol in humans, positive immunological effects and activity as anantioxidant. Isoflavones may also be useful as an alleviator ofvasomotor symptoms in menopausal women, and have been used historicallyin Chinese medicine to treat “hot flashes.”

[0004] Plant proteins also contain significant amounts of phytates,accounting for as much as 85% of the total phosphorus in certain plants.One phytate is phytic acid. Phytic acid is also known as inositolhexaphosphate. As used herein, the term “phytates” means phytic acid andits isomers, the salts and derivatives of phytic acid and its isomers,and/or partially dephosphorylated isomers of phytic acid, and salts andderivatives of partially dephosphorylated isomers of phytic acid. Phyticacid serves several physiological functions and influences thefunctional and nutritional properties of cereals and vegetables by itsability to complex with both proteins and essential minerals. See, e.g.,Ceryan, M., CRC Crit. Rev. Food Sci., Nutr., vol. 13, 297, 1980 andGraf, E., F. Am. Oil Chem. Soc., vol. 60, 1861, 1983. Phytic acid isalso reported to be effective in preventing cancer. See, eg., Reddy, B.S. et al., Cancer Res., vol. 60, no. 17, 2000, pp. 4792-4797;Shamsuddin, A. M. and Vucenik, I., Anticancer Res., vol. 19, no. 5A,1999, pp. 3671-3674. Such properties have prompted research into methodsfor removal of phytates from plant sources, such as soy proteins. U.S.Pat. No. 5,213,835 to Nardelli et al., discloses a process for removingphosphorus from milk and whey proteins.

[0005] Purification of plant proteins and/or isolation of compounds suchas isoflavones and phytates may be achieved by use of such methods asion exchange technology. Such methods are disclosed, for example, inU.S. Pat. Nos. 5,985,338, 5,804,234, and 600,020,471, which are hereinincorporated-by-reference. In such methods, purification of the plantproteins or removal of phytoestrogens or plant estrogens, manganese ornucleotides is effected by passing an aqueous slurry of an isoflavonecontaining material over an active surface such as an anion exchangeresin, thereby binding such compound or compounds to the active surface.As those of skill in the art can appreciate, exchange resins and activesurfaces have a finite capacity, but may be regenerated to an activestate after exhaustion or near-exhaustion. After a certain amount ofuse, reconditioning of the active surface, or ion exchange resin will beuseful. Such regeneration or reconditioning generally comprises removalof the bound compound or compounds from the active surface.

[0006] U.S. Pat. No. 5,804,234 to Suh et al., discloses a method forregenerating or reconditioning exchange resins (or removing the boundcompounds) after contact with plant protein by contact with a saltsolution comprising 6% NaOH, 1% HCl and 1.5% NaHCO3.

[0007] U.S. Pat. No. 6,020,471 to Johns et al., discloses a method forrinsing or releasing bound isoflavones from an ion exchange resin bycontact with an aqueous alcohol solution.

[0008] U.S. Pat. No. 6,146,668 to Kelly et al., discloses anon-chromatographic approach to recovering isoflavones from plantmaterial, and requires the use of organic solvents (e.g., ethyl acetate,hexane, acetone). The residual levels of such organic solvent wouldconstitute a safety concern in the utilization of the recoveredisoflavones in foods for human consumption. Kelly et al. makes noprovision for phytate removal.

[0009] U.S. Pat. No. 6,171,638 to Gugger et al., discloses an ionexchange process for separation and purification of isoflavones,utilizing aqueous alcohol, but makes no provision for phytate removal.

[0010] U.S. Pat. No. 5,789,581 to Matsuura et al., discloses a processfor obtaining certain isoflavones which comprises the use of an aqueousalcohol solution as an eluant.

[0011] U.S. Pat. No. 5,670,632 to Chaihorsky, discloses a process forrecovering isoflavones from a soy extract which comprises the use of ahighly polar sulfonic acid cationic exchange resin as an adsorbent andthe use of an acidic alkanol containing 1 to 3 carbon atoms tofacilitate desorption of the isoflavone 7-glycosides. The solutionutilized in desorption is prepared with a concentrated alcohol (i.e.,96%) to yield a solution of 86% aqueous alcohol. The patent makes noprovision for the recovery of phytates.

[0012] U.S. Pat. No. 5,506,211 to Barnes et al., discloses the use of aparticular isoflavone (genistein) as an inhibitor of osteoclasts. Barneset al. describes the aqueous extraction of isoflavones using 80% aqueousmethanol.

[0013] U.S. Pat. No. 4,428,876 to Iwamura discloses a process forisolating saponins and flavonoids from leguminous plants which comprisesthe use of a polar solvent (such as methanol or aqueous methanol) toelute the saponins and flavonoids adsorbed on a resin.

SUMMARY

[0014] Isoflavones and phytates can be sequentially removed from anactive surface by utilizing a method whereby the active surface(s) arecontacted with an aqueous medium for isoflavone removal and separatelycontacted with an aqueous medium for phytate removal. The aqueous mediumfor isoflavone removal comprises an acidic aqueous alcohol solution. Theaqueous medium for phytate removal comprises either a relativelystronger aqueous acid solution or an aqueous basic solution.Alternatively, for use with some active surfaces, the aqueous medium forphytate removal may comprise an aqueous solution with a pH between about2 and about 7 which is essentially free of alcohol and organic solvents.By this method, isoflavones and phytates are separately and sequentiallyremoved and may be recovered. The recovered isoflavones and phytates maybe utilized in products for human consumption, such as nutritionalproducts or cereals.

DETAILED DESCRIPTION

[0015] Methods are disclosed for sequentially removing isoflavones andphytates from active surface(s) comprising contacting the active surfacewith an aqueous medium for isoflavone removal and separately with anaqueous medium for phytate removal. The aqueous medium for isoflavoneremoval comprises an acidic aqueous alcohol solution. The aqueous mediumfor phytate removal comprises either a relatively stronger (i.e., lowerpH) aqueous acid solution or an aqueous basic solution, or with someactive surfaces may comprise an aqueous solution with a pH between about2 and about 7 which is essentially free of alcohol and organic solvents.

[0016] The acid utilized in the aqueous medium for isoflavone may beselected from various acids, including, but not limited to, acetic acid,citric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoricacid, succinic acid, malic acid, malonic acid, tartaric acid, lacticacid, pyruvic acid, fumaric acid and mixtures thereof. Table I providesa comparison of isoflavone recovery with various acids utilized in theaqueous medium for isoflavone removal. The alcohol(s) utilized in theaqueous medium for isoflavone removal may be selected from variousalcohols, including, but not limited to, methanol, ethanol, propanol andbutanol, and mixtures thereof. Various relative amounts of acid andalcohol will be appropriate for use in the aqueous medium for isoflavoneremoval, depending upon factors such as the strength of the acid and theintended end use of the compound isolated. The amount of alcohol presentin the aqueous medium for isoflavone removal may be between about 10 andabout 90% (v/v). More preferably, the amount of alcohol present isbetween about 50 and about 70% (v/v). Table II provides a comparison ofisoflavone recovery utilizing citric acid in the aqueous medium forisoflavone removal along with varying concentrations of alcohol. Theamount of acid present in the aqueous medium for isoflavone removal willvary according to the strength of the acid (pKa) and concentration ofacid utilized in preparing the medium, but generally should be an amountsufficient for the pH of the aqueous medium to be between about 1.5 andabout 3.5. The amount of acid required to bring the aqueous medium forisoflavone removal to within such a pH range can easily be calculated byone of ordinary skill in the art. Generally amounts between about 0.1and about 40% (w/w) are sufficient. As an example, when glacial aceticacid is utilized in the aqueous medium for isoflavone removal, it ispreferably present in an amount from about 5 to about 40% (v/v), andmore preferably in an amount from about 20 to about 30% (v/v). Table IIIprovides a comparison of isoflavone recovery utilizing an aqueous mediumfor isoflavone removal with varying amounts of ethanol and glacialacetic acid. Table IV provides a comparison of isoflavone recoveryutilizing an aqueous medium for isoflavone removal with 60% ethanol andvarying amounts of glacial acetic acid. When citric acid is utilized inthe aqueous medium for isoflavone removal, it is preferably present inan amount from about 10 to about 40 grams/liter, and more preferably inan amount from about 20 to about 30 grams/liter. A combination of morethan one acid may also be utilized in the aqueous medium for isoflavoneremoval. For example, a suitable aqueous medium for isoflavone removalcould contain approximately 10% glacial acetic acid and approximately 10g/liter citric acid, along with approximately 60% reagent alcohol(reagent alcohol is denatured ethanol; the terms reagent alcohol andethanol are used interchangeably herein). Table V illustratescomparative recovery of isoflavones utilizing a combination of more thanone acid in the aqueous medium for isoflavone removal.

[0017] In another embodiment, the aqueous medium for isoflavone removalcomprises more than one aqueous solution. For example, a first aqueoussolution and a second aqueous solution may be used. The first aqueoussolution may comprise an acid aqueous solution (with a pH between about2 and about 7) essentially free of alcohol, and the second aqueoussolution may comprise an aqueous alcohol solution essentially free ofadded acid. The method of contacting comprises first contacting theactive surfaces with the first aqueous solution, and subsequentlycontacting the active surfaces with the second aqueous solution. Thefirst aqueous solution contains no alcohol or organic solvent, but maycontain at least one acid selected from the group consisting of citricacid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,succinic acid, malic acid, malonic acid, tartaric acid, lactic acid,pyruvic acid, fumaric acid and mixtures thereof. The pH of the firstaqueous solution ranges from about 2 to about 7. More preferably, the pHof the first aqueous solution ranges from about 3 to about 6. The secondaqueous solution may comprise at least one alcohol selected from thegroup consisting of methanol, ethanol, propanol, butanol, and mixturesthereof, but is essentially free of added acid. This second aqueoussolution preferably comprises about 10 to about 90% (v/v) alcohol, morepreferably from about 50 to about 80% (v/v) alcohol.

[0018] Generally, the aqueous medium for phytate removal compriseseither a relatively stronger aqueous acid solution or an aqueous basesolution. Alternatively, as discussed below, when used with some activesurfaces, the aqueous medium for phytate removal may comprise an aqueoussolution with a pH of about 2 to about 7 which is essentially free ofalcohol and organic solvents. The acid utilized in the aqueous mediumfor phytate removal may be selected from various acids, including, butnot limited to acetic acid, citric acid, hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid, succinic acid, malic acid, malonicacid, tartaric acid, lactic acid, pyruvic acid, fumaric acid, andmixtures thereof. Table VI provides a comparison of phytic acid removalwith various acids utilized in the aqueous medium for phytate removal.When the aqueous medium for phytate removal comprises an aqueous basesolution, the base may be selected from various bases including, but notlimited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide,lithium hydroxide, sodium carbonate, and mixtures thereof. For pH valuesin the acidic range (i.e., less than 7), the relative amount of phytatesremoved from an active surface increases with decreasing pH, see TableVII, which illustrates phytic acid and isoflavone recovery at various pHvalues (hydrochloric acid concentrations). The amount of acid or baseutilized in the aqueous medium for phytate removal will vary accordingto the strength of the acid (pKa) or base (pKb), and concentration ofacid or base utilized in preparing the medium, but generally should bean amount sufficient for the pH of the aqueous medium for phytateremoval to be less than about 1, more preferably about 0.1 to about 1.0for an acid solution or alternatively about 13 to about 14 for a basicsolution. The amount of acid or base required to bring the aqueousmedium for phytate removal to within such a pH range can easily becalculated by one of ordinary skill in the art. As an example, whenhydrochloric acid is utilized in the aqueous medium for phytate removal,the amount present is preferably sufficient so that the medium is about0.2 to about 2 M HCl, and more preferably about 0.4 to about 0.8 M HCl.When a base is utilized in the aqueous medium for phytate removal, it ispreferably present in an amount from about 2 to about 10% (w/w), andmore preferably in an amount from about 4 to about 6% (w/w).

[0019] The particular pH that is chosen for both the aqueous medium forisoflavone removal and the aqueous medium for phytate removal may bevaried according to the use which the isolated isoflavones and phytateswill be put. Additionally, the amount of time that the aqueous mediumfor isoflavone removal and the aqueous medium for phytate removal are incontact with the active surfaces and the amount of each medium that isutilized may be varied. Generally, the total amount of isoflavone orphytate recovered increases as the contact time with the active surfacesincreases and as the amount of medium that is utilized increases.However, such increased recovery must be balanced with other factors,including the cost of the protein (isoflavone and phytate source),disposal or treatment costs for additional solvents generated whenincreased volumes of media are utilized, costs for concentrating theisoflavones or phytates, and life time and cost of the active surfaces.In the examples provided below and in the tables provided herein,various contact times and volumes of media are utilized. Volumes ofmedia are expressed herein as bed volumes or column volumes with contacttimes expressed in terms of bed volumes or column volumes per hour orper minute.

[0020] The active surface from which the isoflavones and phytates areremoved and isolated is preferably an anion exchange resin. Suitableanion exchange resins are macroporous resins, preferably a Type I orType II macroporous resin. For anion exchange chromatography, the anionexchange resin is selected from weak base anion exchange resins, strongbase anion exchange resins and mixtures thereof. Representative examplesinclude Amberlite® RA95, IRA-910 and IRA-900 (available from Rohn andHaas Company), Dowex-22 and MSA-1(available from Dow Chemical), andPurolite A510 and A500 (available from Purolite Company). As usedherein, the term resin is meant to include gels, which those skilled inthe art would understand to be useful in the process described herein.Representative gels include Amberlite® IRA 410 (Type II gel, strong baseanion) (available from Rohm and Haas) and IRA 402 (Type II gel, strongbase anion exchange, not macroporous). The anion exchange resin may becontained within a column which has at least one inlet and at least oneoutlet, with the inlet located lower in the column structure than theoutlet. Such a setup allows for a slurry of plant protein to be passedthrough the column and over the resin by entering the column through theinlet and exiting through the outlet. After the desired amount of plantprotein slurry has been passed through the column, the resin is thencontacted with the aqueous medium for isoflavone removal, prepared inthe manner described above and the eluate is collected. After a desiredamount of isoflavone eluate has been collected, the aqueous medium forphytate removal, prepared in the manner described above, may be passedthrough the column and contacted with the resin. The second eluate whichis collected will contain phytates. Table VIII provides a comparison ofthe amounts of isoflavones and phytic acid sequentially recoveredutilizing various combinations of aqueous medium for isoflavone removaland aqueous medium for phytate removal.

[0021] The active surface may also comprise an alkylsilane bonded phasemedium. Alkylsilane bonded phase media are chromatography columnpackings made by chemically bonding an alkylsilane (e.g.,octadecylsilane [ODS or C18] or octylsilane [C8] or butylsilane [C4]) tothe surface of silica. When such are utilized, they are preferablycontained within a column which has at least one inlet and at least oneoutlet, with the inlet located higher in the structure than the outlet.A representative example is Sep-Pak C18, an octadecylsilane, 55-105micrometer diameter, 125 A pore size, 12% carbon (available from WatersCorporation). When the active surface comprises an alkylsilane bondedphase medium, the aqueous medium for phytate removal comprises anaqueous solution with a pH between about 2 and about 7 which isessentially free of alcohol and organic solvents. As used herein, theterm organic solvents includes solvents such as ethanol, methanol,propanol, isopropanol, acetone, dimethylsulfoxide (DMSO),dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, andetc. Suitable solutions for phytate removal for use with an alkylsilanephase bonded medium include, but are not limited to, water and variousbuffers with an appropriate pH. The aqueous medium for phytate removalmay comprise a sufficient amount of an acid selected from the groupconsisting of acetic acid, citric acid, hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid, succinic acid, malic acid, malonicacid, tartaric acid, lactic acid, pyruvic acid, fumaric acid andmixtures thereof in an amount sufficient to the solution to have a pHbetween about 2 and about 7, and is essentially free of alcohol andorganic solvents. The aqueous medium for isoflavone removal for use withan alkylsilane bonded phase medium comprises an acidic aqueous alcoholsolution, as generally described above. The acid utilized in the aqueousmedium for isoflavone may be selected from various acids, including, butnot limited to, acetic acid, citric acid, hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid, succinic acid, malic acid, malonicacid, tartaric acid, lactic acid, pyruvic acid, fumaric acid andmixtures thereof. The alcohol(s) utilized in the aqueous medium forisoflavone removal may be selected from various alcohols, including, butnot limited to, methanol, ethanol, propanol and butanol, and mixturesthereof. Various relative amounts of acid and alcohol will beappropriate for use in the aqueous medium for isoflavone removal,depending upon factors such as the strength of the acid and the intendedend use of the compound isolated. The amount of alcohol present in theaqueous medium for isoflavone removal may be between about 10 and about90% (v/v). More preferably, when utilized with an alkylsilane bondedphase medium, the amount of alcohol present is between about 50 andabout 80% (v/v). As discussed above, the amount of acid present in theaqueous medium for isoflavone removal will vary according to thestrength of the acid (pKa) and concentration of acid utilized inpreparing the medium, but when utilized with an alkylsilane bonded phasemedium generally should be an amount sufficient for the pH of theaqueous medium to be between about 2 and about 7. The amount of acidrequired to bring the aqueous medium for isoflavone removal to withinsuch a pH range can easily be calculated by one of ordinary skill in theart. Generally amounts between about 0.1 and about 40% (w/w) aresufficient. As an example, when glacial acetic acid is utilized in theaqueous medium for isoflavone removal, it is preferably present in anamount from about 5 to about 40% (v/v), and more preferably in an amountfrom about 20 to about 30% (v/v). When citric acid is utilized in theaqueous medium for isoflavone removal, it is preferably present in anamount from about 10 to about 40 grams/liter, and more preferably in anamount from about 20 to about 30 grams/liter. A combination of more thanone acid may also be utilized in the aqueous medium for isoflavoneremoval. Additionally, when the active surface comprises an alkylsilanebonded phase medium, the medium will be contacted first with the aqueousmedium for phytate removal, and then separately contacted with anaqueous medium for isoflavone removal.

[0022] Additionally and alternatively, the active surfaces may compriseboth anion exchange resin and alkylsilane bonded phase medium. When suchare utilized, they are preferably contained within a column which has atleast one inlet and at least one outlet, with the inlet located lower inthe structure than the outlet. Preferably, the alkylsilane bonded phasemedium is positioned closer to the inlet than said anion exchange resin,so that when an aqueous medium is passed through the column it contactsthe alkylsilane bonded phase medium before it contacts the anionexchange resin. Aqueous media for isoflavone removal and for phytateremoval, as described generally above may be utilized. Morespecifically, the aqueous medium for isoflavone removal comprises anacidic aqueous alcohol solution and the aqueous medium for phytateremoval comprises either a relatively stronger (i.e., lower pH) aqueousacid solution or an aqueous basic solution. In an application where bothanion exchange resin and alkylsilane bonded phase medium are utilized asthe active surfaces, the pH of the aqueous medium for isoflavone removalis preferably between about 2 and 3.5.

[0023] Various mixtures of plant protein may be utilized for isolationof isoflavones and phytates. Commercially available mixtures may beutilized or custom blended mixtures may be prepared. One commerciallyavailable plant protein mixture is a soy protein isolate known as ArdexF® (available from Archer Daniels Midland, Inc.). Other materials thatmay be used to supply the source of isoflavones and phytates include anymaterial that contains a detectable level of isoflavones and phytates.Such materials include protein obtainable from soybeans, corn, wheat,peas, beans, cottonseed, peanuts, carrots, alfalfa, algae, potatoes,apples, barley, bluegrass, clovers, coffee, garlic, hops, marijuana,oats, orchard grass, parsley, rice, rye, sage, sesame, yeast, fungus,hydrolyzates thereof, and mixtures thereof.

[0024] Methods described herein may be conducted at room temperature, ormay be conducted at elevated temperatures, such as at about 90° to about120° F. For purposes of this invention, room temperature is defined asbetween about 60° F. and about 80° F. The methods may also be conductedat other temperatures. There is no preference on temperature, althoughas one of ordinary skill in the art will recognize, lower and upperlimits will necessarily be restrained by the freezing points and boilingpoints of the various solutions utilized.

[0025] Processes whereby active surfaces, such as an anion exchangeresin or an alkylsilane bonded phase medium, are utilized to purifyplant proteins will be readily recognizable to those of ordinary skillin the art. Examples of such processes and details regarding suchprocesses are provided in U.S. Pat. Nos. 5,985,338, 5,804,234, and600,020,471. The methods claimed in this patent are not intended to belimited to methods for sequentially removing isoflavones and phytic acidfrom surfaces disclosed or described in those patents.

EXAMPLES

[0026] Isoflavones and phytates can be sequentially isolated by methodswithin the scope of the claims by the following procedures. Theseexamples are being presented as illustrations and should not beinterpreted as limiting in any way.

Example A

[0027] Method Utilizing Various Eluants.

[0028] IEX Resin Preparation: 22 grams of IRA-910 anion exchange resinwere weighed into a 250 milliliter beaker. Using laboratory water (i.e.,purified water (distilled, deionized, demineralized), the resin wastransferred into a 2.5×10 cm glass open column (Bio-Rad catalog no.737-2511) equipped with a 2-way stopcock (Bio-Rad catalog no. 732-8102).(The terms laboratory water, purified water and deionized water are usedinterchangeably herein.) The following solutions were then passedthrough the resin: 45 milliliters of 6% NaOH in approximately 20minutes; followed by 90 milliliters of laboratory water in approximately20 minutes; followed by 100 milliliters of 1% HCl (0.12 M HCl) inapproximately 30 minutes; 180 milliliters of laboratory water inapproximately 40 minutes; 55 milliliters of 1.4% NaHCO₃ in approximately30 minutes; and 360 milliliters of laboratory water in approximately 80minutes.

[0029] IEX Column Preparation: 22.0 grams of Commodity 1922 (PTI soyprotein isolate) were thoroughly suspended in 250 milliliters oflaboratory water in a 400 milliliter beaker. A 1.0-1.5 gram sample wasremoved for isoflavone determination. 22 grams of prepared resin (fromprocedure above) were added to the soy protein isolate suspension andstirred gently for 180 minutes. The resin was allowed to settle to thebottom of the beaker, and another 2.00 milliliter aliquot sample wasremoved for isoflavone determination. The soy protein isolate suspensionwas decanted and discarded. The resin was rinsed with 4×250 millilitersof laboratory water, with decanting and discarding after each addition.Laboratory water was used to pour equal amounts of resin (approximately7 grams) into each of three 2.5×10 cm glass open columns equipped with a2-way stopcock. Each column was rinsed with 20 milliliters of laboratorywater.

[0030] Three eluants were utilized, one for each column, as listed inTable I. The first eluant was 0.48 M HCl in 60% ethanol. The secondeluant was 20% glacial acetic acid in 60% ethanol. The third eluant was20 g/L citric acid in 60% ethanol. Each eluant was passed through one ofthe columns at a rate of 3.6 bed volumes per hour. A total of 14 bedvolumes of eluant were passed through each column. Two fractions werecollected, each consisting of the eluant from 7 bed volumes. The amountsof isoflavones recovered are listed in Table I.

[0031] Isoflavones were determined using gradient elution reverse phaseHPLC. 5.00 milliliters of each fraction were diluted to 25 milliliterswith laboratory water, and tested for isoflavones by the HPLC systemdescribed below:

HPLC System for Isoflavone Determination

[0032] HPLC Column: Waters Nova-Pak C18, 3.9×150 mm, 4 um, 60A, Waters#86344

[0033] Mobile Phase A: 920 mL 0.02M KH₂PO₄, 80 mL acetonitrile; pH 3.1with H₃PO₄

[0034] Mobile Phase B: 400 mL 0.02M KH₂PO₄, 600 mL acetonitrile; pH 3.1with H₃PO₄

[0035] Flow Rate: 0.6 mL/minute

[0036] Column Temperature: 40° C.

[0037] Detection: UV at 262 nm and 250 nm

[0038] Injection: 20 uL

[0039] Run Time: 60 minutes

[0040] Elution Program: Time (minutes) 0 5 40 42 45 48 60 Mobile Phase B(%) 0 0 42 100 100 0 end

Example B

[0041] Method Utilizing Citric Acid and Various Amounts of Ethanol.

[0042] IEX resin and columns were prepared as described above forExample A except that the soy protein isolate and resin suspension wasstirred for 2 hours, and 5 columns were prepared, and each columncontained 4.4 grams of resin. For this example, five eluants containingcitric acid and varying amounts of ethanol were prepared and utilized.All eluants contained 20 g/L citric acid. The amounts of ethanolutilized in the five eluants were: 0% (v/v), 10% (v/v), 20% (v/v), 60%(v/v) and 90% (v/v).

[0043] As in Example A, each eluant was passed through one of thecolumns at a rate of 3.6 bed volumes per hour. A total of 14 bed volumesof eluant were passed through each column. Each eluate was tested forisoflavones according to the method described above in Example A. Theamounts of isoflavones recovered are listed in Table II.

Example C

[0044] Method Utilizing Various Amounts of Glacial Acetic acid, Ethanoland Water.

[0045] IEX Resin Preparation: The IEX Resin was prepared as described inExample A above.

[0046] IEX Column Preparation: 4.0 grams of soy protein isolate (PTI #C7H-XTO-9001) were thoroughly suspended in 200 milliliters of laboratorywater in a 400 milliliter beaker. A 2.00 milliliter aliquot sample wasremoved for isoflavone determination (“column feed”). 20 grams ofprepared resin (from procedure above) were added to the soy proteinisolate suspension and stirred at approximately 400 rpm for 60 minutes.The resin was allowed to settle to the bottom of the beaker, and another2.00 milliliter aliquot sample was removed for isoflavone determination(“unbound by IEX resin”). The soy protein isolate suspension wasdecanted and discarded. The resin was rinsed with 2×200 milliliters oflaboratory water, with decanting and discarding after each addition.Laboratory water was used to resuspend the resin, and equal amounts ofresin (approximately 3.3 grams) were poured into each of six 20milliliters columns. Each column was rinsed with 10 milliliters oflaboratory water. 15.0 milliliters of eluant was transferred by pipetinto the column, and eluted at a flow rate of approximately 1 columnvolume per minute. A total of 5 column volumes of eluate were passedthrough each column. The eluate was tested for isoflavones according tothe method described above in Example A.

[0047] Various eluants were prepared and utilized as listed in TableIII. The eluants contained varying amounts of ethanol, water and glacialacetic acid. The amounts of isoflavones removed are listed in Table III.

Example D

[0048] Method Utilizing Ethanol and Varying Amounts of Glacial AceticAcid.

[0049] IEX resin and columns were prepared as described above forExample B. For this example, five eluants containing ethanol and varyingamounts of glacial acetic acid were prepared and utilized. All eluantscontained 60% ethanol (v/v). The concentrations of glacial acetic acidpresent in the five eluants were: 0% (v/v), 10% (v/v), 20% (v/v), 30%(v/v), and 40% (v/v).

[0050] Each eluant was passed through one of the columns at a rate ofapproximately 10 column volumes per hour. A total of 10 bed volumes ofeluant were passed through each column. The eluates were collected andeach eluate was tested for isoflavones according to the method describedabove in Example A. The amounts of isoflavones recovered are listed inTable IV.

Example

[0051] Method Utilizing Various Eluants.

[0052] IEX Resin and columns were prepared as described above forExample A.

[0053] For this example, three eluants were prepared and utilized. Thefirst eluant contained 20 g/L citric acid in 60% ethanol. The secondcontained 20% glacial acetic acid in 60% ethanol. The third contained 10g/L citric acid and 10% glacial acetic acid in 60% ethanol. Each eluantwas passed through one of the columns at a rate of approximately 3.6 bedvolumes per hour. A total of 14 bed volumes was passed through eachcolumn. The eluates were collected and tested for isoflavones. Theamounts of isoflavones recovered are listed in Table V.

Example F

[0054] Method Utilizing Various Eluants.

[0055] IEX Resin and columns were prepared as described above in ExampleA, except that 6 columns were prepared, each column containing 3.6 gramsof resin.

[0056] For this example, six eluants were prepared and utilized. Thefirst contained 20% glacial acetic acid in 60% ethanol. The secondcontained 20% glacial acetic acid in water. The third contained 20 g/Lcitric acid in 60% ethanol. The fourth contained 20 g/L citric acid inwater. The fifth contained 0.48 M HCl in 60% ethanol. The sixthcontained 0.48 M HCl in water. Each eluant was passed through one of thecolumns at a rate of approximately 3.6 bed volumes per hour. A total of14 bed volumes was passed through each column. The eluates werecollected and each eluate was tested for phytic acid. The amounts ofphytic acid recovered are listed in Table VI.

[0057] Phytic Acid was determined by the following procedure. Forethanolic eluate samples, 7.00 milliliters of the eluate was evaportedto dryness with compressed N₂, and the residue resuspended in 7.00milliliters of 0.02 M NaMalonate (pH 2.5). For aqueous eluate samples,the pH was adjusted to 2.5 with NaOH. 3.00 milliliters of eluate samplewas transferred by pipet into each of two 1-dram vials. A phytasesuspension was prepared by throughly suspending 36 mg of phyrate (Sigmacatalog no. P-9792; crude, from Aspergillus ficum) in 4 milliliters of0.02 M NaMalonate (pH 2.5). To the first vial was added 400 microlitersof 0.02 M NaMalonate; this was the phorphorus control. To the secondvial was added 400 microliters of the phytate suspension; this was thephytase digest. The vials were capped, mixed well, and incubated at 40°C. for 120 minutes. The vials were removed from the water bath. Thesample suspensions were filtered through a 0.45 micrometer Acrodiscsyringe filter (Gelman P/N 4497). The filtrate was tested for inorganicphosphorus, using Sigma's colorimetric Inorganic Phosphorus test kit(Sigma catalog no. 670-A) and a spectrophotometer. Eluate phytic acidwas calculated by subtracting the non-phytic acid phosphorus (asmeasured in the phosphorus control) from the phytase digest phosphorusand by using the appropriate dilution factor and molecular weights forphosphorus (30.97) and for phytic acid (660.0).

Example G

[0058] Method Utilizing Ethanol and Varying Amounts of HCl.

[0059] IEX Resin Preparation: The IEX Resin was prepared as described inExample A above.

[0060] IEX Column Preparation: 22 grams of Commodity 1922 (PTI soyprotein isolate “PP1610”) were thoroughly suspended in 250 millilitersof laboratory water in a 400 milliliter beaker. A 1.0-1.5 gram samplewas removed for isoflavone determination. 22 grams of prepared resin(from procedure above) were added to the soy protein isolate suspensionand stirred gently for 120 minutes. The resin was allowed to settle tothe bottom of the beaker, and another 2.00 milliliters aliquot samplewas removed for isoflavone determination. The soy protein isolatesuspension was decanted and discarded. The resin was rinsed with 250milliliters of laboratory water and decanted and the supernatantdiscarded. Rinsing was repeated until the resin was thoroughly rinsed ofvisible soy protein isolate solids. An equal quantity of resin(approximately 4.4 grams) was transferred into each of five 2.5×10 cmglass open columns (Bio-Rad # 737-2511) equipped with a 2-way stopcock(Bio-Rad #732-8102). Each column was rinsed with 50 milliliters oflaboratory water. At this point, elution of isoflavones and/or phytateswith desired eluants may begin.

[0061] For this example, five eluants were prepared and utilized. Allcontained 60 milliliters of ethanol and 40 milliliters of HCl in varyingconcentrations. HCl concentrations were 1.25 N, 1.00 N, 0.75 N, 0.50 Nand 0.25 N. The entire volume of each eluant was passed through one ofthe columns at a rate of approximately 25 milliliters per hour. Eacheluant was collected and analyzed for isoflavones, phytic acid andprotein. The amounts of isoflavones, phytic acid and protein recoveredare listed in Table VII.

[0062] Phytic acid was determined by pipetting 8.00 milliliters ofcolumn eluate into a 30 milliliter beaker. Ethanol was evaporated with astream of compressed nitrogen. 15.0 milliliters of 0.05 M malonic acidwere added to the beaker. The pH was adjusted to 2.5 with 2 N NaOH. Thesample was diluted to 25 milliliters with 0.05 M malonic acid. 10.0milliliters was pipetted into each of two vials. 5 mg of phytase (EC3.1.3.8, Sigma # P-9792, 3.5U/mg solid) was added to one of the twovials. Both vials were incubated at 37° C. for fourteen hours. The vialswere cooled to room temperature and 10.0 milliliters of 20%trichloroacetic acid (w/v) was added to each vial. The vials werecapped, mixed well and 3-4 milliliters were filtered through a 0.45micrometer membrane (Gelman Acrodisc, P/N 4497). 1.00 milliliters offiltrate was tested for inorganic phosphorus by Sigma Test Kit 670-A(inorganic phosphorus; colormetric endpoint method; Sigma 1999 Catalog).

[0063] Protein was determined by pipetting 5.00 milliliters of columneluate into a 2 dram vial. The sample was evaporated to dryness with astream of compressed nitrogen. The residue was suspended in 2milliliters of 6 M HCl and transferred into a 2 milliliter ampule. Theampule was nitrogen-blanketed, flame-sealed and heated at 110° C. for 22hours. The ampule was cooled to room temperature and the sampleevaporated to dryness. The residue was reconstituted in 2.00 millilitersof Beckman Na-S buffer and tested for amino acids on a Beckman Model6300 Automated Amino Acid Analyzer (i.e., by ion exchangechromatography, post-column ninhydrin derivatization, and visibleabsorbance detection).

Example H

[0064] Method Utilizing Sequential Eluants.

[0065] IEX Resin and columns were prepared as described above in ExampleA.

[0066] For this example, three different sequential eluants wereutilized, with varying amounts of each sequential eluant passed throughthe particular column. In the first column, three different eluants wereutilized sequentially. The first eluant was 20% glacial acetic acid in60% ethanol; 14 bed volumes of this eluant were utilized. The secondeluant was 20% glacial acetic acid in water; 14 bed volumes of thiseluant were utilized. The third eluant was 0.48 M HCl in water; twofractions of 20 bed volumes each were utilized.

[0067] In the second column, three different eluants were utilizedsequentially. The first eluant was 20 g/L citric acid in 60% ethanol; 14bed volumes of this eluant were utilized. The second eluant was 20 g/Lcitric acid in water; 14 bed volumes of this eluant were utilized. Thethird eluant was 0.48 M HCl in water; two fractions of 20 bed volumeseach were utilized.

[0068] In the third column, two different eluants were utilizedsequentially. The first eluant was 0.48 M HCl in 60% ethanol; 14 bedvolumes of this eluant were utilized. The second eluant was 0.48 M HClin water; two fractions were utilized, the first of 14 bed volumes andthe second of 20 bed volumes.

[0069] All eluants were passed through the columns at approximately 3.6bed volumes per hour. Eluates were collected and isoflavones and phyticacid measured by the procedures described above (Examples A and F). Theamounts of isoflavones and phytic acid recovered are listed in TableVIII.

Example I

[0070] Method Utilizing an Alkylsilane Active Surface with VariousEluants.

[0071] Seven cartridges, each packed with 100 mg of octadecylsilane(C18) bonded phase medium, were prepared by conditioning each with 20bed volumes of methanol, and then rinsing each with 20 bed volumes oflaboratory water. Onto each cartridge was loaded 1.00 milliliters of a1% (w/w) slurry of soy protein isolate in laboratory water.

[0072] In this experiment, seven different eluants were prepared andutilized. The eluants were as follows: water; 20% glacial acetic acid inwater; 20 g/L citric acid in water; ethanol; 20% glacial acetic acid in60% ethanol; 20 g/L citric acid in 60% ethanol; and 10% glacial aceticacid and 10 g/L citric acid in 60% ethanol. Ten bed volumes of eluantwere passed through each cartridge at a rate of four bed volumes perminute. Eluates were collected, and isoflavones measured by theprocedure described above (Example A). The isoflavone recoveries arelisted in Table IX.

Example J

[0073] Method Utilizing Glacial Acetic Acid and Ethanol in the AqueousMedium for Isoflavone Removal and Hydrochloric Acid in the AqueousMedium for Phytate Removal.

[0074] From a well-stirred slurry of soy protein isolate (2.0 g of soyprotein isolate in 100 milliliters of laboratory water) are removed 2milliliters for control testing of the isoflavone and phytate contents.To the remainder of the suspension are added 10.0 g of anion exchangeresin (IRA-910 in the chloride form). The whole is stirred magneticallyfor about 60 minutes, whereupon the suspension is decanted from theanion exchange resin beads. The latter are rapidly rinsed twice withlaboratory water (100 milliliters) by decanting, and then slurried witha further 100 milliliters of laboratory water and transferred to a glasscolumn chromatography tube (2.5×10 cm) equipped with a support frit andan outlet valve. The resin is rinsed with a further 100 milliliters ofwater, the eluant flow rate being adjusted to 0.5 column volumes/minute.Two hundred milliliters of an eluant (the aqueous medium for isoflavoneremoval) composed of ethanol, glacial acetic acid, and laboratory waterin the ratio by volume of 3:1:1 are passed through the resin bed at 0.5column volumes/minute. The column eluate is collected and tested forisoflavones by gradient elution reverse phase HPLC. Two hundredmilliliters of an eluant (the aqueous medium for phytate removal)composed of 0.48M hydrochloric acid in deionized water are passedthrough the resin bed at 0.5 column volumes/minute. The column eluate iscollected and tested for phytic acid by an enzymatic/colorimetric method(the enzyme phytase is used to selectively release phosphate from phyticacid, and the phosphate is then quantified as inorganic phosphorus bythe phosphomolybdate colorimetric method).

Example K

[0075] Method Utilizing Citric Acid and Ethanol in the Aqueous Mediumfor Isoflavone Removal and Sulfuric Acid in the Aqueous Medium forPhytate Removal.

[0076] The procedure is followed as set forth in Example 1 except thatthe aqueous medium for isoflavone in Example J is replaced with 20 g/Lcitric acid in 60% (v/v) ethanol, and the aqueous medium for phytateremoval is replaced with 0.24M sulfuric acid in deionized water.

Example L

[0077] Method Utilizing Hydrochloric Acid and Ethanol in the AqueousMedium for Isoflavone Removal and Sodium Hydroxide in the Aqueous Mediumfor Phytate Removal.

[0078] The procedure is followed as set forth in Example J, except thatthe aqueous medium for isoflavone removal in Example J is replaced with0.05M hydrochloric acid in 60% (v/v) ethanol, and the aqueous medium forisoflavone is replaced with 5% (w/v) sodium hydroxide in deionizedwater. The flow rate (for both aqueous media) is also changed to 2column volumes/minute.

Example M

[0079] Method Utilizing C18 Bonded Phase Medium.

[0080] From a well-stirred slurry of soy protein isolate (2.0 g of soyprotein isolate in 100 milliliters of laboratory water) is removed 2milliliters for control testing of the isoflavone and phytate contents.A cartridge containing 100 mg of a C18 bonded phase medium isconditioned with 2 column volumes of ethanol and two column volumes oflaboratory water. One milliliter of the soy protein isolate suspensionis passed through the C18 bonded phase medium at a flow rate of 1 columnvolume/minute. The column is rinsed with one milliliter of laboratorywater. The column eluate (i.e., from the load+the rinse) is collectedand tested for phytic acid by an enzymatic/colorimetric method (asdescribed above). Two milliliters of an eluant composed of 20 g/L ofcitric acid in 60% ethanol are passed through the cartridge at a flowrate of 1 column volume/minute. The column eluate is collected andtested for isoflavones by gradient elution reverse phase HPLC.

[0081] Particular embodiments have been described above that fall withinthe scope of the invention as set forth in the claims. These embodimentsare not intended to limit the scope of the invention to the specificforms disclosed. The invention is intended to cover all modificationsand alternative forms falling within the spirit and scope of theinvention. TABLE I Isoflavone Recovery Comparison Isoflavone Recovery (%of IEX Bound) Fraction 1 Fraction 2 Total IEX* Eluant Composition (7BV)** (7 BV)** (14 BV)** 0.48 M HCl in 60% ethanol 46 18 64 20% glacialacetic acid in 60% 46 16 62 ethanol 20 g/L citric acid in 60% ethanol 3626 62

[0082] TABLE II Isoflavone Recovery vs. Eluant Alcohol Concentration -Isoflavone Elution from IRA-910 IEX Column. Isoflavone Recovery (% IEXColumn Eluant Composition** of IEX Bound) 20 g/L citric acid monohydratein 0% ethanol (v/v) 7 20 g/L citric acid monohydrate in 10% ethanol(v/v) 12 20 g/L citric acid monohydrate in 20% ethanol (v/v) 19 20 g/Lcitric acid monohydrate in 60% ethanol (v/v) 64 20 g/L citric acidmonohydrate in 90% ethanol (v/v) 49

[0083] TABLE III Isoflavone Removal vs. Eluant Composition Isoflavones*Removed by 5 CV** at 1 Eluant Composition CV/minute (% of (percentagesare volume basis) IEX Bound) 50% ethanol, 20% glacial acetic acid, 30%water 46.6 60% ethanol, 20% glacial acetic acid, 20% water 49.4 70%ethanol, 20% glacial acetic acid, 10% water 38.3 80% ethanol, 20%glacial acetic acid, 0% water 18.4 60% ethanol, 30% glacial acetic acid,10% water 41.0 60% ethanol, 40% glacial acetic acid, 0% water 39.7

[0084] TABLE IV Isoflavone Recovery vs. Eluant Acetic Acid Isoflavonesin 10 Isoflavone Eluant Composition CV* Recovery** 60% ethanol, 0%glacial acetic acid  5.0 mg/L 9.3% 60% ethanol, 10% glacial acetic acid24.7 mg/L 46.2% 60% ethanol, 20% glacial acetic acid 29.3 mg/L 54.8% 60%ethanol, 30% glacial acetic acid 28.8 mg/L 53.95% 60% ethanol, 40%glacial acetic acid 20.6 mg/L 38.4%

[0085] TABLE V Isoflavone Recovery from IRA-910 IEX Column IsoflavoneRecovery (% IEX Column Eluant Composition* of IEX Bound) 20 g/L citricacid monohydrate in 60% ethanol 55 20% glacial acetic acid in 60%ethanol 56 10 g/L citric acid in l0% glacial acetic acid in 53 60%ethanol

[0086] TABLE VI Phytic Acid Removal Comparison mg of Phytic Acid removedIEX Eluant Composition from IEX Column* 20% glacial acetic acid in 60%ethanol <1 20% glacial acetic acid in water <1 20 g/L citric acid in 60%ethanol <1 20 g/L citric acid in water <1 0.48 M HCl in 60% ethanol 190.48 M HCl in water 30

[0087] TABLE VII IEX Elution vs. Hydrochloric Acid Concentration IEXEluant HCl Conc. pH* Isoflavones Phytic Acid Protein A 0.5 N 0.30 857 μg22.7 mg 11.6 mg B 0.4 N 0.40 903 μg 15.8 mg 11.9 mg C 0.3 N 0.52 820 μg9.35 mg 10.2 mg D 0.2 N 0.70 904 μg 5.61 mg 10.9 mg E 0.1 N 1.00 805 μg2.53 mg  9.5 mg

[0088] TABLE VIII Sequential Elution of Isoflavones and Phytic Acid IEXIsoflavone Phytic Col- # of Recovery (% Acid umn Eluant Composition BV*of IEX Bound) (mg) A 20% glacial acetic acid in 60% 14 62 <1 ethanol 20%glacial acetic acid in water 14 — <1 0.48 M HCl in water 20 — 50 0.48 MHCl in water 20 — 24 B 20 g/L citric acid monohydrate 14 62 <1 in 60%ethanol 20 g/L citric acid monohydrate 14 — <1 in water 0.48 M HCl inwater 20 — 47 0.48 M HCl in water 20 — 22 C 0.48 M HCl in 60% ethanol 1464 19 0.48 M HCl in water 14 — 30 0.48 M HCl in water 20 — 20

[0089] TABLE IX Isoflavone Recovery from C18 Bonded Phase Medium -Eluant Recovery Comparison Isoflavone Recovery Eluant (10 bed volumes)(% of C18 Bound) Water <1% 20% glacial acetic acid in water 30% 20 g/Lcitric acid in water <1% ethanol 96% 20% glacial acetic acid in 60%ethanol 89% 20 g/L citric acid in 60% ethanol 92% 10% glacial aceticacid, 10 g/L citric acid in 60% 95% ethanol

What is claimed is:
 1. A method for the sequential removal ofisoflavones and phytates from one, or more, active surfaces, comprisingthe steps of: a) providing active surfaces comprising an anion exchangeresin; b) contacting said active surfaces with an aqueous medium forisoflavone removal; and c) contacting said active surfaces with anaqueous medium for phytate removal.
 2. A method as defined in claim 1,wherein said active surfaces are contained within a column which has atleast one inlet and at least one outlet, said inlet located lower in thecolumn than said outlet, such that said aqueous medium for isoflavonemay be contacted with said active surfaces and a first eluate collected,and then said aqueous medium for phytate removal may be contacted withsaid active surfaces and a second eluate collected.
 3. A method asdefined in claim 2, wherein said first eluate contains less than 1%(w/v) of phytate.
 4. A method as defined in claim 2, wherein said methodis conducted at room temperature.
 5. A method as defined in claim 2,wherein said method is conducted at a temperature from 90° to 120° F. 6.A method as defined in claim 2, wherein said aqueous medium forisoflavone removal comprises: a) 1-40% (w/w) of at least one acidselected from the group consisting of acetic acid, citric acid, malicacid, malonic acid, lactic acid, and mixtures thereof; and b) 10-90%(v/v) of at least one alcohol selected from the group consisting ofmethanol, ethanol, propanol, butanol, and mixtures thereof.
 7. A methodas defined in claim 2, wherein said aqueous medium for isoflavoneremoval comprises 50-70% v/v of at least one alcohol selected from thegroup consisting of methanol, ethanol, propanol, butanol, and mixturesthereof.
 8. A method as defined in claim 2, wherein said aqueous mediumfor isoflavone removal comprises: (a) at least one acid selected fromthe group consisting of acetic acid, citric acid, hydrochloric acid,sulfuric acid, nitric acid, phosphoric acid, succinic acid, malic acid,malonic acid, tartaric acid, lactic acid, pyruvic acid, fumaric acid andmixtures thereof; (b) 10-90% (v/v) of at least one alcohol selected fromthe group consisting of methanol, ethanol, propanol, butanol, andmixtures thereof; and has a pH ranging from 1.5 to 3.5.
 9. A method asdefined in claim 2, wherein said aqueous medium for phytate removal hasa pH less than
 1. 10. A method as defined in claim 2, wherein saidaqueous medium for phytate removal comprises at least one acid selectedfrom the group consisting of acetic acid, citric acid, hydrochloricacid, sulfuric acid, nitric acid, phosphoric acid, succinic acid, malicacid, malonic acid, tartaric acid, lactic acid, pyruvic acid, fumaricacid, and mixtures thereof, and has a pH less than
 1. 11. A method asdefined in claim 2, wherein said aqueous medium for phytate removal hasa pH between 13 and
 14. 12. A method as defined in claim 2, wherein saidaqueous medium for isoflavone removal comprises at least two separateaqueous solutions.
 13. A method for the sequential removal ofisoflavones and phytates active surfaces, comprising the steps of: a)providing active surfaces comprising alkylsilane bonded phase media; b)contacting said active surfaces with an aqueous medium for isoflavoneremoval; and c) contacting said active surfaces with an aqueous mediumfor phytate removal.
 14. A method as defined in claim 13, wherein saidalkylsilane bonded phase medium is contacted with an aqueous medium forphytate removal which comprises an aqueous solution with a pH of 2 to 7,essentially free of alcohol and organic solvents, and said alkylsilanebonded phase medium is then contacted with an aqueous medium forisoflavone removal.
 15. A food containing isoflavones isolated by amethod as defined in claim
 2. 16. A food containing phytates isolated bya method as defined in claim
 2. 17. A method for sequentially isolatingisoflavones and phytates from plant materials comprising: a) providingat least one anion exchange resin; b) providing a slurry of plantprotein that contains isoflavones and phytic acid; c) placing the anionexchange resin in a structure which has at least one inlet and at leastone outlet, and said inlet is located lower in the structure than theoutlet; d) passing the slurry into the structure and over the resin byentering through the inlet and exiting through the outlet; e) contactingsaid resin with an aqueous medium for isoflavone removal and collectinga first eluate; and (f) then contacting the resin with an aqueous mediumfor phytate removal and collecting a second eluate.